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Figure Lgends
Figure 1. Expression of the fusion protein prM-E-△-PA3 in the baculovirus expression system. (A) Schematic diagram of the fusion protein prM-E-△-PA3 fragment. “△” represents a deletion of the stem-transmembrane (ST-TM) region. ZIKV prM-E gene: the prM-E gene of ZIKV (accession: KX601168.1; does not contain the start codon). ZI-△-PA gene: the sequences from the N-terminus to the C-terminus were prM-E-△ of ZIKV, a linker and protein anchor 3 (PA3). ZI-JE-△-PA gene: the signal peptide of ZI-△-PA gene was replaced with the signal peptide of JEV. ZI-GP-△-PA gene: the signal peptide of ZI-△-PA gene was replaced with the gp67 signal peptide. (B) IFA detection of the ZIKV E protein (100×). Recombinant baculovirus-infected Sf9 cells were subjected to IFA for the detection of ZIKV E protein expression. Uninfected Sf9 cells were used as a cell control. Positive results showed bright green fluorescence, and negative results showed red due to Evans blue. (C) WB detection of the ZIKV E protein in baculovirus-infected cells. The proteins were detected in the supernatant or the sonicated supernatant of infected cells. M represents the protein marker, and the lines in the figure correspond to 100, 70 and 55 kDa.
Figure 2. Investigation of fusion-GEM-complexes binding. (A) Combination diagram of the prM-E-△-PA3 protein binding to GEM. (B) Electron microscopy observation of fusion-GEM-complexes. The scale bars represent 200 nm. GEM was used as the control. (C) IFA detection of ZIKV E protein in fusion-GEM-complexes (1000×). (D) WB detection of the ZIKV E protein in fusion-GEM-complexes. M represents the protein marker.
Figure 3. Efficacy of the immunogen in BALB/c mice. (A) Experimental scheme. BALB/c mice (n=18 mice/group) were immunized with ZI-△-PA-GEM, ZI-JE-△-PA-GEM or ZI-GP-△-PA (10 µg) mixed with a complex adjuvant of ISA201 VG and poly(I:C) by intramuscular injection. Mice injected with PBS combined with adjuvant were used as the control. On the 3rd, 6th and 9th days after the first immunization, lymphocytes from the inguinal lymph nodes of the mice (3 mice/group/day) were analyzed. Nine mice were boosted twice at 2-week intervals, and sera were collected on days 0, 28, 42, 56 and 70. On the 35th day, splenic lymphocytes from the mice (n=3 mice/group) were analyzed. (B) ZIKV-specific IgG titers in the serum (dilution 128-fold) were measured by ELISA at the indicated time points, displayed as the OD450 ratio of sample/control. (C) ZIKV neutralizing antibody titers detected at week 6 by a standard 50% plaque reduction neutralization test (PRNT50). (D) The ratios of IgG2a/IgG1 at week 6 were determined by ELISA.
Figure 4.Detection of B cell and DC activation early after immunization. On the 3rd, 6th and 9th days after the first immunization, lymphocytes from the inguinal lymph nodes of the mice (3 mice/group/day) were analyzed by flow cytometry. (A) B cell activation, displayed as CD19+ CD40+. (B) Representative flow cytometric plots of lymphocytes of B cell activation on D6. (C) DC activation, displayed as CD11c+MHCII+. (D) Representative flow cytometric plots of lymphocytes of DC activation on D6. The data are expressed as the mean ±SD for each group. *p<0.05; **p<0.01; ***p<0.001.
Figure 5. Splenocyte proliferation analysis. On the 35th day after immunization, splenic lymphocytes from the mice (3 mice/group) were harvested and stimulated with purified ZIKV E protein. (A) After ex vivo culture for 44 h, the proliferative index was determined using a CCK-8 assay. (B) After culture for 24 h, the levels of IFN-γ and IL-4 secreted by the splenocytes were quantified by the ELISpot assay. (C) After culture for 72 h, the cytokine levels in splenocyte culture supernatants were measured by MesoScale Discovery (MSD). The data are expressed as the mean ±SD for each group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 6. Lymphocyte activation analysis.On the 35th day after immunization, splenic lymphocytes from the mice (3 mice/group) were stimulated with purified ZIKV E protein and cultured ex vivofor 72 h. The activation of CD4 T cells (A-B), CD8 T cells (C-D) and B cells (E-F) was measured by flow cytometry. (A, C, E) Representative flow cytometric plots of lymphocytes from each group. (B, D, F) Percentages of the indicated lymphocytes. The data are expressed as the mean ±SD for each group. *p<0.05; **p<0.01; ***p<0.001.
Figure 7. Central memory T cells (TCMs) analysis. On the 35th day after immunization, splenic lymphocytes from the mice (3 mice/group) were stimulated with purified ZIKV E protein and cultured ex vivo for 72 h. The proportion of TCMs among CD4+ T (A-B) or CD8+ (C-D) T cells was evaluated by the presence of CD44 and CD62L surface markers. (A, C) Representative flow cytometric plots of lymphocytes from each group. (B, D) Percentages of the indicated lymphocytes. The data are expressed as the mean ±SD for each group. *p<0.05; **p<0.01.